Analytical characterization and stability evaluation of high-purity melittin isolated from crude bee venom via...

Melittin (MEL), the principal peptide component of honeybee venom (Apis mellifera), is of considerable interest owing to its broad biological activities. However, the isolation of MEL in high purity remains analytically challenging, primarily due to the chemical complexity of crude venom and the amphipathic nature of the peptide. Here, we describe a one-step, scalable reversed-phase liquid chromatography (RP-HPLC) approach that affords MEL with 99.05 % recovery and 98.44 % purity, markedly exceeding the quality of available commercial standards. The identity and structural integrity of the purified peptide were confirmed through complementary analytical techniques, including RP-HPLC, electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and circular dichroism (CD) spectroscopy. Accelerated stability testing under ICH conditions (40°C, 75 % RH) over six months demonstrated a substantially higher chemical and conformational stability of purified MEL compared with crude venom, with degradation following first-order kinetics. Taken together, these findings establish an efficient and reproducible chromatographic workflow for the preparative isolation and analytical characterization of MEL from a complex natural source, while stability profiling highlights its suitability for downstream pharmaceutical applications.